Euglobulin-based method for determining the biological activity of defibrotide

ABSTRACT

It is disclosed a method for determining the biological activity of defibrotide, which comprises the steps of: a) bringing into contact defibrotide, mammalian euglobulin and a substrate specific for the plasmin which, by reaction with the plasmin, provides a measurable product; and b) measuring the amount of product formed at successive times, to thereby determine the biological activity of the defibrotide. Liquid defibrotide formulations are also disclosed, preferably water solutions, having a defined biological activity and, in particular, having an activity of 25 to 35 IU/mg of defibrotide, preferably from 27 to 32 IU/mg and, more preferably, from 28 to 32 IU/mg.

This application is a continuation of U.S. application Ser. No.16/816,741, filed Mar. 12, 2020, which is a continuation of U.S.application Ser. No. 15/844,801 filed Dec. 18, 2017, which is acontinuation of U.S. application Ser. No. 14/408,272 filed Dec. 15,2014, now U.S. Pat. No. 9,902,952, and which is a 371 of internationalPCT/IT2012/000193, filed Jun. 22, 2012, each of which is hereinincorporated by reference in its entirety.

The present invention relates to a method for determining the biologicalactivity of defibrotide and, more especially, relates to an indirectenzymatic method for determining the biological activity of defibrotide.

TECHNICAL FIELD OF THE INVENTION

Defibrotide (Merck Index, 1996, no. 2915) is a substance of naturalorigin which is obtained by extraction from animal organs and which isconstituted by the sodium salt of polydeoxyribonucleotides having a lowmolecular weight. Defibrotide has been the subject of numerouspharmacological investigations which have suggested that it be appliedin therapy as an anti-thrombotic agent (U.S. Pat. No. 3,829,567).

In addition, defibrotide has also been used successfully in thetreatment of peripheral arteriopathies, in acute renal insufficiency(U.S. Pat. No. 4,694,134) or in acute myocardial ischaemia (U.S. Pat.No. 4,693,995).

Defibrotide is currently undergoing clinical trials to be used for thetreatment and prevention of venous occlusive disease (VOD).

Like other biological substances which are obtained by extraction,defibrotide is also subject to a limited variability of compositionwhich is typical of natural biopolymers. A classical example of thissituation is provided by heparin whose variability from batch to batchin terms of chain length, molecular weight, composition, degree ofsulphatation, etc. is well known. The consequence of this is that thesame amounts by weight of defibrotide could in fact be non-equivalentfrom the point of view of a specific biological activity.

The process of extraction, isolation and purification cannot per seensure absolute reproducibility of the product, precisely owing to itsintrinsic biopolymeric nature.

However, if well controlled, it is possible to reduce this variability:for that purpose, studies have been made of standardized industrialprocesses for isolating defibrotide by extraction from organs, such as,for example, that described in U.S. Pat. No. 4,985,552.

The product obtained according to the above-mentioned process ischaracterized by the determination of some specific physico-chemicalparameters, such as, for example, electrophoretic mobility, thecoefficient of extinction, optical rotatory power and mass-averagerelative molecular mass. However, those parameters depend basically onthe structure of defibrotide and are not capable of providinginformation on the biological activity thereof.

As far as we know, the only methods that have been reported to be usedhitherto to evaluate the biological activity of defibrotide are thefibrin plate test and the thromboelastographic recording of theeuglobulin lysis time [Prino G., Mantovani M., Niada R., Coccheri S.,Butti A., Indagini preliminari sull'attivita fibrinolitica, nell'animalee nell'uomo, di una nuova sostanza presente in diversi organi animali,Simposio Internazionale: La ricerca scientifica nell'industriafarmaceutica in Italia, Rome, 2-4 Oct. 1975-11 Farmaco, Ed. Prat.)(1969), 24, 552-561] and the method based on plasmin disclosed in U.S.Pat. No. 7,338,777, herein incorporated by reference.

However, the above-mentioned method thromboelastographic recording ofthe euglobulin lysis time is characterized by considerable experimentalcomplexity, by unsatisfactory reproducibility and precision and, in thespecific case of thromboelastographic recording, by a response linearitylimited to very restricted concentration ranges.

As to the plasmin-based method, the enzymatic activity of plasmin isnormally determined by various standard in vitro tests. One of the mostcommonly used methods is the determination by spectrophotometry orfluorimetry of the chromogenic or fluorogenic compounds that are freedby the action of plasmin on suitable substrates [Haemostasis, (1978), 7,138-145]. Peptide substrates having the formula A₁-A₂-A₃-X are generallyused in which A₁ and A₂ are amino acids that are predominantlynon-polar, A₃ is lysine or arginine and X represents the measurablefreed compound, for example para-nitroaniline (pNa) or 2-naphthylamine(NA) [Haemostasis, (1978), 7, 146-149]. In addition to theabove-mentioned peptide substrates, success has been achieved usingother, simpler, compounds, such as, for example,p-nitrobenzyl-p-toluenesulphonyl-L-arginine [Haemostasis, (1978), 7,105-108].

The rate at which the compound X is released into the incubation mediumis proportional to the activity (Units/mg) of plasmin present in thesample. The method disclosed in U.S. Pat. No. 7,338,777 is thus based onthe finding that, in the plasmin-evaluation tests described above,defibrotide increases the rate of release of compound X proportionallyto its concentration.

However, such a method is conducted in TRIS buffer without any otherplasma/serum activator/inhibitor. Therefore, the procedure does notreflect the physiological condition nor accurately simulates themechanism of action of defibrotide in vivo.

Hitherto, therefore, no truly valid, precise and reproducible methodshave been described and validated for determining the biologicalactivity of defibrotide reflecting in an accurate way the mechanism ofaction of the product in a complex biological system (in vivo).

We have developed a simple and reliable method for determining thebiological activity of defibrotide, which enables the samples obtainedby extraction to be controlled and therefore enables medicinalpreparations based on defibrotide to be standardized.

The method to which the present invention relates enables the specificbiological activity of defibrotide to be determined in comparison with areference standard with a high degree of precision and accuracy.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a plot showing the kinetics of release of pNA from thesubstrate S-2251, by means of mammalian euglobulin fraction which isactivated and non-activated with defibrotide (concentration 0-50 μg/ml,0-100 min).

FIG. 2 is a plot illustrating the sigmoid that arises in relation to astandard and test sample of defibrotide.

FIG. 3 is a plot showing the “absorbance versus time” of the standardpreparations (ex.: S1_Ca, S1_Cb, S1_Cc) identify a suitable linear range(ex.: from 30 to 35 min).

DESCRIPTION OF THE INVENTION

The present invention therefore relates to a method for determining thespecific biological activity of samples of defibrotide, which methodcomprises the steps of:

-   a) bringing into contact defibrotide, euglobulin and a substrate    specific for the plasmin which, by reaction with the plasmin,    provides a measurable product and-   b) measuring the amount of product formed at successive times.

The method of the invention is an indirect in vitro method fordetermining the activity of defibrotide, which is based on thefunctional interactions between defibrotide and euglobulin.

Euglobulin is that fraction of serum globulin that is insoluble indistilled water but soluble in saline solutions.

Euglobulin contains fibrinogen, PAI-1, tissue plasminogen activator(tPA), plasminogen, and to a lesser extent alpha 2-antiplasmin and alsofactor VIII.

The present inventors have surprisingly found that defibrotide catalyzesthe hydrolysis of plasminogen into plasmin. Consequently, whendefibrotide is incubated with euglobulin and a substrate specific forplasmin, such as a peptide of formula A₁-A₂-A₃-X as disclosed byHaemostasis, (1978), 7, 138-149, herein incorporated by reference, therate at which the compound X is released into the incubation mediumincreases proportionally to the concentration of defibrotide itself.

In other terms, defibrotide catalyzes the hydrolysis of plasminogencontained in euglobulin into plasmin; which plasmin enzymatically reactswith the substrate specific for plasmin, preferably a cromogenicsubstrate, to provide a measurable product.

The method of the present invention thus further comprises the steps of:c) determining the rate of release of the measurable product during thecourse of the enzymatic reaction of both a standard sample and a testsample of defibrotide; d) correlating, mathematically and/orgraphically, the rate of release with the corresponding defibrotideconcentration to obtain the biological activity of the test sample ofdefibrotide.

The defibrotide sample used for the determination according to thepresent invention is generally prepared by extraction from organs inaccordance with known procedures, such as described, for example, inU.S. Pat. No. 4,985,552 which has already been mentioned and which isalso herein incorporated by reference.

A batch of normal industrially manufactured defibrotide was chosen asthe reference sample (standard) and was used to prepare the calibrationcurves in accordance with the method of the present invention.

In general, the present method provides precise and accuratemeasurements of defibrotide even in the presence of contaminants, suchas, for example, RNA, heparin, degraded defibrotide (defibrotide fromwhich purin or pyrimidine has been removed) or ethanol, provided theyare in concentrations, generally less than 10% by weight, such as not toimpair the system.

In addition to permitting the determination of defibrotide, the methodalso allows the determination of other biologically equivalentsubstances derived from defibrotide, such as, for example, deaminateddefibrotide or, more simply, defibrotide denatured/degraded bycombination of physicochemical conditions.

The present method is sufficiently sensitive to detect concentrations ofdefibrotide lower than or equal to 2.5 μg/ml (final concentration in thedetermination system) and, generally, expresses good correlation up tomaximum concentration values higher than or equal to 1000 μg/ml.

The euglobulin used is generally any mammalian euglobulin fraction, suchas, for example, bovine, porcine, rabbit ‘or human euglobulin, with apreference for human and bovine euglobuline.

However, although euglobulin fraction is the enzymatic system of choice,the use of other equivalent enzymatic systems, such as, for example,diluted plasma and serum (up to 1:10 with buffers), artificially createdor isolated combinations of plasminogen, tPA, uPA, PAI-1&2 alpha 2antiplasmin and the like enzymatic systems which are chemically andbiologically related and have similar functionality, falls within thescope of the present invention.

In the method of the present invention, the substrate for the plasminmay be understood as being any substrate specific for plasmin which,under the conditions of the method, frees a detectable hydrolysisproduct X.

Depending on the nature of the detectable group X, alternative systemsof detection commonly known to the person skilled in the art can beadopted equally well. Spectrophotometric or fluorimetric detectionsystems are particularly advantageous, especially spectrophotometricsystems.

The substrates generally used are ones that are specific forplasminoge-plasmin assay. It is preferable to use peptides of theformula A₁-A₂-A₃-X, in which A₁ and A₂ are amino acids that arepredominantly non-polar, A₃ is lysine or arginine and X is thedetectable group. Examples of those substrates are Val-Leu-Lys-pNa,Val-Phe-Lys-pNa or pyroGlu-Phe-Lys-pNa in which the group X detectableby spectrophotometry is para-nitroaniline (pNA). Other suitablesubstrates, for example Val-Gly-Arg-2NA, contain 2-naphthylamine, whichis measurable by fluorimetry. A particularly preferred substrate is thecompound H-D-Valyl-L-Leucyl-L-Lysine-p-nitroaniline(H-D-Val-Leu-Lys-pNA).

The specific substrates used for determining defibrotide activity ineuglobulin fraction are generally commercially available.

The determination method of the present invention is carried out byplacing defibrotide sample in euglobulin solution, at a specific pH andmolarity.

In particular, euglobulin fractions obtained from mammalian plasma arereconstituted dissolving and diluting the euglobulin to the originalvolume of the generating plasma with saline buffer (ex.: the quantity ofeuglobulin fraction obtained from 10 mL plasma are dissolved andreconstituted to 10 mL with saline buffer at pH between 7 and 8).

However, as regards of the substrate concentrations of from 0.3 to 4 mM,preferably from 2.5 to 3.5 mM and advantageously of 3 mM, are generallyused in the case of a chromogenic substrate, while concentrations offrom 0.05 to 0.15 mM are used in the case of a fluorogenic substrate.

The determination method of the invention, like other enzymatic methods,is sensitive to the pH of the medium.

In fact, it cannot generally be applied at extreme pH values where theenzymatic system would be inactivated.

It is also preferable for the pH of the medium not to undergo variationat any time during the period when measurements are being taken, andtherefore euglobuline fraction is reconstituted with buffer systemsselected from those normally used for biological tests. Suitable buffersystems may be, for example, phosphate buffer, citrate buffer ortris(hydroxymethyl)aminomethane hydrochloride and sodium chloride(TRIS-NaCl) buffer. The reconstitution of the euglobuline fraction ispreferably carried out with TRIS-NaCl.

In the present method it is usually preferred to maintain the pH of themedium in a range of approximately from 7 to 8, more preferably atapproximately 7.4-7.6.

In addition, it is preferred to maintain the concentration of the buffersystem in a range of from 10 to 200 mM, preferably at approximately 50mM. More specifically for the TRIS-NaCl the concentrations should be 50mM for TRIS and 150 mM for sodium chloride

The method of the invention for determining defibrotide biologicalactivity, defibrotide sample solutions is diluted directly intoeuglobulin fraction, then the chromogenic or fluorogenic substrate isadded. In particular, in order to enable the measurements it ispreferable to preliminary dilute/dissolve defibrotide in TRIS-NaClbuffer in order to obtain a mother stock solution of both, sample andstandard. From the mother stock solutions the sample and the standardare diluted, by serial dilution, into defined volume of euglobulinfraction till the analytical concentration range which is about 1 to1000 μg/mL of defibrotide

An important parameter in the present method of determination is thetemperature. It is preferable for the same temperature to be maintainedthroughout the entire duration of the measurements and for all of thesamples determined, both as regards the construction of the referencecurves and during the measuring stage. To that end, it is preferable touse temperature controlled apparatus and also, where necessary, it ispossible to proceed with several sets of measurements, changing theposition of the samples appropriately in order to ensure that the systemhas maximum thermal homogeneity.

Generally, this method of determination is applied in a temperaturerange of, for example, from 25 to 40° C., preferably from 35 to 39° C.,and even more preferably at 37° C.

According to the present invention, measurement of the concentration ofcompound X released in the medium by the action of defibrotide startswhen all of the reagents have been added and continues for apredetermined time and at a predetermined frequency as a function of thechemical nature of X and of the detection system.

Similarly to other methods of biological determination, the method ofthe invention also provides for a calibration stage and a measuringstage which are preferably carried out in the same microplateplate inorder to reduce as far as possible the incidence of experimentalvariability.

The calibration stage comprises the acquisition of the absorbance datarelating to the samples at known increasing concentrations ofdefibrotide (standard), the statistical reprocessing of those data andthe extrapolation of calibration curves, which express the correlationbetween the increase in the rate of the enzymatic reaction of theinvention and the concentration of defibrotide present in the euglobulinfraction. In the measuring stage, owing to the correlation obtained inthe calibration stage, it is possible to determine the unknownbiological activities of samples of defibrotide on the basis of theabsorbance values measured and processed under the same conditions.

In more detail, the experimental protocol generally provides for thepreparation of several samples, both standard and unknown, at variousknown concentrations of defibrotide. The defibrotide samples areprepared by progressive dilution of the mother solutions in accordancewith a predetermined dilution factor.

In the present method, it is preferred to prepare at least 5concentrations of the standard and 5 concentrations of the sample to betested, preparing at least 4 replicates for each concentration of thestandard and, similarly, for each concentration of the test sample,generally for successive 1:2 dilutions of mother solutions.

Both the standard and test-sample concentrations of defibrotide aregenerally from 0.1 to 1000 μg/ml.

The concentrations of the test sample are preferably of the same orderof magnitude as the concentrations of the standard.

In accordance with the above illustration, the measurements for eachconcentration are preferably carried out on 1 microplates where theposition of each sample, the standard and the test sample, respectively,at corresponding concentration is preferably alternated. According tothis scheme for the arrangement of the samples, which is explained inmore detail in the experimental part, for each concentration of bothstandard and test-sample defibrotide, at least 4 absorbance values aremeasured for each time.

The set of measurements described above are taken at predeterminedtimes, that is to say, first of all at time to, that is to say, when allof the components have been added, before the enzymatic reaction of theinvention has started, and subsequently at precise intervals and for aperiod of time sufficient to acquire the necessary data.

Preferably, the absorbance measurements are continued up to a maximum of90 minutes, with readings taken every 1-10 minutes. More advantageously,the readings are taken every minute. The photometric absorbance readingsare performed at a wavelength which depends on the nature of thedetectable group X freed in the course of the enzymatic hydrolyticreaction. In the specific case in which X is p-NA, the absorbance ismeasured at 405 nm.

The absorbance readings of the standard and unknown defibrotide samples,known as raw data, generally originate directly from the same apparatusthat provides for the reading operation; they are tabulated in such amanner that an absorbance value is expressed for each time and well.

The raw data are then processed, using, for example, the SpreadSheet—Microsoft Excel®. This first processing operation leads to thecalculation of the average absorbance and of the associated standarddeviation, at each time and for each set of readings, each setcomprising at least 4 replicates for each concentration of both standardand test-sample defibrotide.

Further statistical processing of the data is carried out withcommercially available software for biological assay determination asdescribed by Finney D J, Statistical Method in Biological Assay, 2nd ed.Ch. Griffin, London and relevant Pharmacopoeias.

To be more precise, according to the present invention, defibrotidebiological assay determination can be performed using parallel linemodel, slope ration model and four-parameter logistic curve models asdefined, for example, by the relevant European Pharmacopoeia Generaltext 5.3, Statistical Analysis”

As illustrated in FIG. 1, by placing the time on the abscissa and theabsorbance on the ordinate, straight lines will be obtained whose slope“b” will be proportional to the rate of enzymatic reaction: byincreasing the concentration of defibrotide, the rate of hydrolysis and,proportionally, the value of “b” will increase. Finally, the slopevalues, calculated as described above for each set of replicates ofstandard defibrotide and test-sample defibrotide, are correlated withthe concentration of defibrotide to which they relate. Suitablemathematical transformation of the abscissa (i.e. log of defibrotideconcentration) can be used in lieu of the real value.

Graphically, that correlation gives rise to a sigmoid for the standardand a sigmoid for the test sample (FIG. 2); the central portions of thesigmoid have two straight lines which are generally parallel and thedistance between which is a function of the difference in biologicalactivity between the test sample and the standard. This interval is usedfor potency determination using the parallel line model as described byFinney D J, Statistical Method in Biological Assay, 2nd ed. Ch. Griffin,London.

For a more specific determination, the four-parameter logistic curvemodels is used and in this case the entire sigmoid curve of both, sampleand standard, is used for the calculation of the relative biologicalpotency of the sample.

In a preferred embodiment of the present invention, the standardsolutions and the solutions of the samples of defibrotide to bedetermined are introduced into the respective wells of the microplate.The euglobulin fraction are prepared at the moment of use and it is thedilution media of defibrotide stock solution. Finally, the solutioncontaining the chromogenic substrate is added. The microplate is thenplaced in the thermostated reader and, after rapid agitation, readingsof the system's absorbance are taken at predetermined intervals and forthe predetermined period of time. The raw data obtained are thenprocessed, thus determining the unknown activities of the defibrotidesamples.

As it shall be appreciated from the following examples, the methodaccording to the present invention allows to obtain liquid defibrotideformulations, preferably water solutions, having a defined biologicalactivity and, in particular, having an activity of 25 to 35 IU/mg ofdefibrotide, preferably from 27.5 to 32.5 IU/mg and, more preferably,from 28 to 32 IU/mg.

Liquid defibrotide formulations are preferably marketed in form ofcontainers, more preferably vials, containing 200 mg of defibrotide in2.5 ml of buffered water solution (preferably at a pH from 6.5 to 8.5,more preferably from 7 to 8), to be diluted before use; consequently,when the biological activity is assessed with the method of the presentinvention, each container presents a defibrotide activity of 5000 to7000 IU, preferably 5500 to 6500 IU, more preferably 5600 to 6400.

Those and other aspects of the invention will be better illustrated inthe following examples which are not, however, to be regarded aslimiting the invention.

Examples

The following materials were used in the examples given here:

Apparatus

Main features: Microplate reader for UV-Vis absorbance determinationequipped with thermostatic chamber and absorbance filter at 405 nm.Detection Kinetic Absorbance determination at 405 nm Plate Type 96-wellclear for UV-Vis Determination Chamber 37° C. Total Absorbance 45-60 minTemperature recording time Absorbance About 1 × min recording frequency

Programs & Software

-   -   Microsoft Excel® (Microsoft Corporation, Redmond, Wash., USA)

Substances

-   -   Defibrotide (Gentium)    -   Chromogenic substrate S-2251 (Chromogenix Instrumentation        Laboratory S.p.A., Milan, Italy)    -   Tris(hydroxymethyl)aminomethane (TRIS)-NaCl, (Sigma-Aldrich,        Milan, Italy)    -   1N HCl (Carlo Erba reagenti, Milan, Italy)    -   1N NaOH (Carlo Erba reagenti, Milan, Italy)    -   Bovine Plasma (Tebu Bio Italia, Magenta (MI), Italy)    -   Glacial Acetic Acid (Carlo Erba reagenti, Milan, Italy)

Solutions

-   -   TRIS-NaCl (1 L Preparation): Into a 1 L beaker quantitatively        transfer 6.06 g of TRIS-HCl and 2.2 g of NaCl. Dissolve in 500        mL of purified water and adjust the pH to 7.4 with about 40 mL        of HCl 1N. Quantitatively transfer the solution into a        volumetric flask of 1 L and dilute the solution to volume with        purified water. Store the solution into refrigerator (2-8° C.)    -   Chromogenic Substrate S2251 (CAS 63589-93-5) 3 mM (15.2 mL        Preparation): Dissolve about 25 mg of chromogenic substrate with        15.2 mL of purified water. Store the solution into refrigerator        (2-8° C.)    -   Bovine euglobulins (10 mL Preparation). In a container with a        minimum capacity of 300 mL introduce 240 mL of ice-cooled        purified water and under stirring add 10 mL bovine plasma.        Adjust the pH to 5.3±0.1 with acetic acid 1%. Allow to settle at        2-8° C. for 1 to 16 hours. Remove the clear supernatant solution        by siphoning and collect the precipitate by centrifugation at        2.800 rpm for 5 minutes and 4° C. Suspend the precipitate        dispersing mechanically (e.g.: by means of a laboratory glass        rod) in 5 mL of ice-cooled purified water, shake for about 5 min        and collect the precipitate by centrifugation at 2.800 rpm for 5        minutes and 4° C. Disperse the precipitate mechanically into        about 10 mL of TRIS-NaCl; to facilitate the dissolution of the        precipitate crush the particles of the precipitate with a        suitable instrument (es.: laboratory glass rod). Store the        obtained suspension at 2-8° C. for not less than 1 hour and not        more than 6 hours before its use.

Standard and Sample Defibrotide Solutions

Reference Stock Solution

Into 20 mL volumetric flask quantitatively transfer about 100 mg ofdefibrotide reference standard accurately weighed. Dissolve the powderwith about 10 mL TRIS-NaCl and bring the volume with the same solvent.Dilute 1:4 the obtained solution with TRIS-NaCl in order to obtain adefibrotide RS solution of about 1.25 mg/mL.

Sample Stock Solution

Into 20 mL volumetric flask quantitatively transfer about 100 mg ofdefibrotide sample accurately weighed. Dissolve the powder with about 10mL TRIS-NaCl and bring the volume with the same solvent. Dilute 1:4 theobtained solution with TRIS-NaCl in order to obtain a defibrotide samplesolution of about 1.25 mg/mL.

Reference and sample solutions preparation

-   -   a) Defibrotide 125 ug/mL: dilute 1:10 defibrotide stock solution        (Reference and Sample) with TRIS NaCl (corresponding to 50 ug/mL        into the plate well). Into an eppendorf tube quantitatively        transfer 500 uL of the prepared solution and mix with 500 uL of        euglobulin. Close the tube and store in ice-cooled water.    -   b) Defibrotide 62.5 ug/mL: dilute 1:2 solution (a) with TRIS        NaCl (corresponding to 25 ug/mL into the plate well), Into an        eppendorf tube quantitatively transfer 500 uL of the prepared        solution and mix with 500 uL of euglobulin. Close the tube and        store in ice-cooled water.    -   c) Defibrotide 31.25 ug/mL: dilute 1:2 solution (b) with TRIS        NaCl (corresponding to 12.5 ug/mL into the plate well). Into an        eppendorf tube quantitatively transfer 500 uL of the prepared        solution and mix with 500 uL of euglobulin. Close the tube and        store in ice-cooled water.    -   d) Defibrotide 12.5 ug/mL: dilute 1:2.5 solution (d) with TRIS        NaCl (corresponding to 5 ug/mL into the plate well). Into an        eppendorf tube quantitatively transfer 500 uL of the prepared        solution and mix with 500 uL of euglobulin. Close the tube and        store in ice-cooled water.

Blank Solution

Mix 1 volume of euglobulins with 1 volume of TRIS NaCl solution (ex.:500 uL+500 uL)

Plate Deposition

According to the proposed deposition scheme (see Table 1 below) add ineach well of the plate 200 uL of standard or sample or blank solution.Note different deposition scheme can be used according to theavailability and configuration of automatic pipettes. However not lessthan 4 depositions for each reference and sample solution must be usedfor the assay.

Immediately before incubation of the plate into the microplate readeradd in each well 50 uL of Chromogenic substrate.

TABLE 1 1 2 3 4 5 6 7 8 9 10 11 12 A — BLK S1_Ca S1_Cb S1_Cc S1_Cd — — —— — — B — BLK S2_Ca S2_Cb S2_Cc S2_Cd — — — — — — C — BLK S3_Ca S3_CbS3_Cc S3_Cd — — — — — — D — BLK S4_Ca S4_Cb S4_Cc S4_Cd — — — — — — E —BLK U1_Ca U1_Cb U1_Cc U1_Cd — — — — — — F — BLK U2_Ca U2_Cb U2_Cc U2_Cd— — — — — — G — BLK U3_Ca U3_Cb U3_Cc U3_Cd — — — — — — H — BLK U4_CaU4_Cb U4_Cc U4_Cd — — — — — —S1, S2, S3, S4: Reference Solution deposition 1, deposition 2,deposition 3, deposition 4, U1, U2, U2, U5: Sample solution deposition1, deposition 2 deposition 3, deposition 4, Ca, Cb, Cc etc.: Defibrotidereference and sample concentration a, b, c etc.

BLK Blank Solution Calculation and Results

From the kinetic plot “absorbance versus time” of the standardpreparations (ex.: S1_Ca, S1_Cb, S1_Cc) identify a suitable linear range(ex.: from 30 to 35 min, see FIG. 3).

Identification of the linear kinetic range (A@405 nm vs time).

Calculate for each preparation of the standard and of the sample theresponse of the assay (Slope) in the pre-defined time range as follow.

${{{Sample}\&}\mspace{14mu}{Standard}\mspace{14mu}{Response}} = {\frac{A_{b} - A_{a}}{T_{b} - T_{a}} \times 1000}$

Where:

-   Aa is the Absorbance values at Time Ta (30 min from the plot above)-   Ab is the Absorbance value at Time Tb (35 min from the plot above)

Report the obtained value in a tabular format as reported in table 2.

TABLE 2 Concentration Level Standard Preparation Sample Preparation[ug/mL] S1 S2 S3 S4 U1 U2 U3 U4  5 S1_Cd S2_Cd S3_Cd S4_Cd U1_Cd U2_CdU3_Cd U4_Cd 12.5 S1_Cc S2_Cc S3_Cc S4_Cc U1_Cc U2_Cc U3_Cc U4_Cc 25S1_Cb S2_Cb S3_Cb S4_Cb U1_Cb U2_Cb U3_Cb U4_Cb 50 S1_Ca S2_Ca S3_CaS4_Ca U1_Ca U2_Ca U3_Ca U4_Ca

Plot the responses for the substance to be examined and for the standardagainst the logarithms of the concentration and calculate the activityof the substance to be examined using the parallel line model as definedby the relevant 5.3.2 chapter of the Ph. Eur Current edition. Not lessthan 3 consecutive serial dilutions of the reference and of the sampleshould be used (e.g., defibrotide concentration 5 μg/mL, 12.5 μg/mL, 25μg/mL, 50 μg/mL, or 5 μg/mL, 12.5 μg/mL, 25 μg/mL, or 12.5 μg/mL, 25μg/mL, 50 μg/mL).

Analysis of the Variance

The analysis of the variance is performed according to the section5.3.2.3 of the Ph. Eur. current edition and Finney D J (1964)Statistical Method in Biological Assay 2nd ed.

Test for Validity

-   -   1) The linear regression terms is significant, i.e. the        calculated probability is less than 0.05. If this criterion is        not met, it is not possible to calculate 95% C.I.    -   2) The Term of non-parallelism is not significant, i.e. the        calculated probability is less than 0.05.    -   3) The term for non linearity is not significant, i.e. the        calculated probability is less than 0.05.

Acceptance Criteria

-   -   4) The estimated potency is not less than 90% and not more than        111% of the stated potency.    -   5) The confidence limits (P=0.95) of the estimated potency are        not less than 80% and not more than 125% of the stated potency

Calculation

${{Sample}\mspace{14mu}{Potency}\mspace{14mu}\left( {{UI}\text{/}{mg}} \right)} = \frac{R \times {Std}\mspace{14mu}{Potency}\mspace{14mu}\left( {{UI}\text{/}{mg}} \right) \times {{Conc}.{Std}}\mspace{14mu}\left( {{mg}\text{/}{mL}} \right)}{{{Conc}.{Sample}}\mspace{14mu}\left( {{mg}\text{/}{mL}} \right)}$

where:

-   -   R: Result of sample obtained by the parallel line model analysis    -   Std Potency: Stated potency of the Standard (UI/mg on dry        substance)    -   Conc. Std: Concentration of the Standard (mg/mL on dry        substance)    -   Conc. Sample: Concentration of the Sample (mg/mL on dry        substance)

Assay Applied to Defibrotide Formulations

The above-disclosed assay has been used to determine the biologicalactivity of liquid formulations containing 200 mg of defibrotide in 2.5ml (80 mg per ml) and having the quali-quantitative composition reportedin table 3.

TABLE 3 Reference to Standard 200 mg Concentration Component QualityFunction Injection per mL Defibrotide In-house Drug 200.00 mg 80.00 mgstandard Substance Sodium Citrate, USP-EP Buffer  25.00 mg 10.00 mgDihydrate Water-for- USP-EP Solvent/ q.s. to 1.00 mL injection vehicle2.5 mL Sodium NF-EP pH q.s. to — hydroxide adjustment 6.8-7.8 1M orhydrochloric acid 1M Nitrogen NF-EP Inert gas q.s. — to displace the air

The results are reported in table 4.

TABLE 4 Potency Potency Batch IU/mg IU/container of 200 mg 688 31 6200738 34 6800 785 32 6400 836 30 6000 0406 31 6200 DS0617  35 7000DV0502* * Not available DV0601  35 7000 1070010073 32 6400 1080010016 326400 1080020018 31 6200 1080030021 31 6200 1080040110 32 6400 108005011435 7000 1080060117 34 6800 Mean 33 Min 30 Max 35 RSD (%) 5, 4 *Used asReference Standard

1. A defibrotide formulation consisting of defibrotide, sodium citrate,and water for injection, having a potency of 25 to 35 IU/mg, and aconcentration of at least 80 mg/mL, wherein the defibrotide potency isdetermined by a method comprising the steps of: a) bringing into contactdefibrotide, a biological composition comprising plasminogen, and asubstrate specific for plasmin which, by reaction with plasmin, providesa measurable product; and b) measuring the amount of product formed atsuccessive times, to thereby determine the potency of the defibrotide.2. The defibrotide formulation of claim 1, wherein the formulation has apotency of 27.5 to 32.5 IU/mg.
 3. The defibrotide formulation of claim2, wherein the formulation has a potency of 28 to 32 IU/mg.
 4. Thedefibrotide formulation of claim 1, wherein the formulation is a watersolution.
 5. The defibrotide formulation of claim 4, wherein theformulation has a pH of from 6.5 to 8.5.
 6. The defibrotide formulationof claim 5, wherein the formulation has a pH of from 7 to
 8. 7. Thedefibrotide formulation of claim 1, wherein the biological compositioncomprising plasminogen is a human, rabbit or bovine composition.
 8. Thedefibrotide formulation of claim 1, wherein plasmin which reacts withthe substrate specific for plasmin is released by the plasminogen. 9.The liquid defibrotide formulation of claim 1, wherein the substratespecific for the plasmin is a chromogenic substrate.
 10. The defibrotideformulation of claim 1, wherein the substrate specific for the plasminis a compound of formula A1-A2-A3-X in which A1 and A2 are non-polaramino acids, A3 is lysine or arginine and X is the measurable product.11. The defibrotide formulation of claim 10, wherein the measurableproduct X is selected from the group consisting of para-nitroaniline and2-naphthylamine.
 12. The defibrotide formulation of claim 10, whereinthe substrate specific for plasmin isH-D-Valyl-L-Leucyl-L-Lysine-p-nitroaniline.
 13. The defibrotideformulation of claim 10, wherein the measurable product X is measured byspectrophotometry or spectrofluorimetry.
 14. The defibrotide formulationof claim 1, wherein the mammalian composition comprising plasminogen isobtained from a volume of plasma and reconstituted to the same volume ofthe originating plasma or diluted up to 1:10 with suitable buffer andthe substrate specific for the plasmin is a chromogenic/fluorogenicsubstrate having a concentration of from 2.5 to 3.5 mM.
 15. Thedefibrotide formulation of claim 1, wherein said method is carried outin a reaction medium which is an aqueous solution buffered to a pH offrom 7 to
 8. 16. The defibrotide formulation of claim 1, wherein themethod is maintained at a temperature of from 35 to 39° C.
 17. Thedefibrotide formulation of claim 1, wherein the substrate specific forplasmin has a concentration of from 0.3 to
 4. 18. The defibrotideformulation of claim 1, wherein the method comprises the steps of: c)determining the rate of release of the measurable product during thecourse of the reaction of both a standard sample and a test sample ofdefibrotide; d) correlating the rate of release with the correspondingdefibrotide concentration to obtain the potency of the test sample ofdefibrotide.
 19. The defibrotide formulation of claim 17, wherein theconcentration of the substrate specific for plasmin is 3 mM.